The invention relates to fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) is a single molecule detection method that has been used to detect molecules in small volume samples, e.g., femtoliters. FCS is a technique that employs confocal optics to limit the volume of sample studied to that of a confocal plane. The confocal plane is defined by the optical components of the system. FIG. 1 illustrates a laser beam illuminated volume of sample. A box is shown superimposed on the waist of the laser beam. The optical components of the system function to position the confocal plane in the waist of the laser beam. The diffusion of fluorescently labeled particles into and out of the illuminated confocal volume generates data related to fluorescence intensity fluctuation. Information about the particles in the sample can be extracted from this data.
Optics are employed in FCS to reduce out-of-focus light and limit image detection to the desired focal plane of in-focus light (i.e., the sample plane, which is also referred to as the image plane). The excitation volume (i.e., confocal volume) is minimized by illuminating the volume with a laser beam that has been focused to the limit of the resolution of a high numerical aperture objective lens. Out-of-focus light is eliminated by placing a field pinhole (i.e., aperture) in a conjugate image plane of the objective lens and before the detector. Eliminating out-of-focus light limits the volume of detection to the plane in which the object is focused. Signals from planes either above or below the object plane are focused either above or below the conjugate image plane, which renders them inefficiently collected due to the location of the aperture through which the focused image is allowed to pass. Fluorescently labeled particles that are in the sample of interest are detected only when the particles are present at the image plane of the confocal volume.
FCS is a technique used in biophysics, biochemistry, and cell biology. FCS can be used to study events at the level of single molecules. The diffusion times and the interaction of macromolecules, the absolute concentration of fluorescently labeled particles and the kinetics of chemical reactions can be measured using FCS. Applications of FCS have included studies related to ligand-receptor binding, protein-protein and protein-DNA interactions, and the aggregation of fluorescently labeled particles. The theory and applications of FCS are described in various references including Rigler, R. (1995), “Fluorescence correlations, single molecule detection and large number screening. Applications in biotechnology,” J Biotechnol 41(2-3): 177-86; Schwille, P. (2001), “Fluorescence correlation spectroscopy and its potential for intracellular applications.” Cell Biochem Biophys 34(3): 383-408; and Hess, S. T., S. Huang, et al. (2002). “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41(3): 697-705.
Typical FCS instruments are configured to include an epifluorescence microscope or a confocal microscope. These configurations are relatively large and require a user to focus and adjust the microscope and align the various components of the instrument prior to use.